Macs mitochondria isolation kit

Manufactured by Miltenyi Biotec

The MACS Mitochondria Isolation Kit is a laboratory equipment product designed for the efficient isolation of mitochondria from various cell and tissue types. The kit provides a reliable and standardized protocol for the separation and purification of mitochondria, enabling researchers to study the structure, function, and dynamics of these organelles.

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Lab products found in correlation

Citrate synthase assay kit, by Merck Group (1 mentions)

Close spelling variants of this product

MACS isolation kit (27 citations) Mitochondria Isolation Kit (25 citations) Isolation kits (10 citations) MACS kits (8 citations)

3 protocols using macs mitochondria isolation kit

Assessing Mitochondrial Integrity by Citrate Synthase

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Mitochondria isolated using the MACS Mitochondria Isolation Kit (Miltenyi) were examined for intactness using a citrate synthase assay kit (Sigma-Aldrich) according to manufacturer’s instructions. Briefly, isolated mitochondria from approximately 10⁷ HeLa cells were incubated at 37°C for 0 minutes or 6 hours, then were separated into two equal aliquots, one in which mitochondria were left intact, and one in which mitochondria were lysed. Because citrate synthase activity is restricted to the mitochondrial matrix, inner mitochondrial membrane intactness can be assessed by comparing the citrate synthase activity in a sample of intact mitochondria to that of a sample of lysed mitochondria using an indicator dye detected spectrophotometrically. This mitochondrial intactness assay indicated that, after 6 hours of incubation in mitochondrial secretion assay buffer, the percent of mitochondria that were ruptured rose from 19.0% at 0 minutes to 29.5% at 6 hours (data not shown). These data indicate that mitochondria isolated in our laboratory are largely intact, even after several hours.

Clay H.B., Parl A.K., Mitchell S.L., Singh L., Bell L.N, & Murdock D.G. (2016). Altering the Mitochondrial Fatty Acid Synthesis (mtFASII) Pathway Modulates Cellular Metabolic States and Bioactive Lipid Profiles as Revealed by Metabolomic Profiling. PLoS ONE, 11(3), e0151171.

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Isolation of Mitochondria from HeLa Cells

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Mitochondria from HeLa cells were harvested using the MACS Mitochondria Isolation Kit (Miltenyi) according to manufacturer’s protocol. Briefly, approximately 10⁷ HeLa cells were washed and lysed, then incubated with magnetic beads linked to an anti-TOM22 antibody, which binds the outer surface of the mitochondria. The mitochondria-linked beads were passed through a column in a magnetic field, retaining the beads and mitochondria. Upon removal from the magnetic field, isolated mitochondria were then flushed from the column.

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Efficient Extraction of Cellular and Mitochondrial DNA

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For extraction of total cellular DNA, cells were washed once with PBS before being solubilised in cell lysis buffer (75 mM NaCl, 50 mM EDTA, 20 mM HEPES (pH 7.8), 0.5% SDS, 0.2 mg/ml proteinase K) and added to an equal volume of phenol. DNA was purified by two consecutive rounds of sequential extraction with phenol and chloroform, followed by isopropanol precipitation and resuspension in TE pH 8.0. For preparation of mtDNA for EM analysis, mitochondria were first isolated from 1 × 10⁷ cells using a MACS mitochondria isolation kit (Miltenyi Biotec) according to manufacturer’s instructions. Cells were lysed by 15 passages through a 27 gauge needle fitted to a 1 mL syringe, and nuclei were removed by centrifugation at 300 × g for 10 min at 4°C. The supernatant from this step was applied to the column through a 20 μm pre-separation filter. Isolated mitochondria were lysed by the addition of 0.5% SDS and 0.5 mg/ml proteinase K at 55°C for 2 hr. DNA was then extracted by phenol/chloroform extraction and ethanol precipitation, and pellets were resuspended in TE pH 8.0.

Nicholls T.J., Nadalutti C.A., Motori E., Sommerville E.W., Gorman G.S., Basu S., Hoberg E., Turnbull D.M., Chinnery P.F., Larsson N.G., Larsson E., Falkenberg M., Taylor R.W., Griffith J.D, & Gustafsson C.M. (2017). Topoisomerase 3α Is Required for Decatenation and Segregation of Human mtDNA. Molecular cell, 69(1), 9-23.e6.

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